Although interactions of metallic nanoparticles (NPs) with various microorganisms have been previously explored, few studies have examined how metal sensitivity impacts NP toxicity. The present study investigated the effects of copper NPs (Cu-NP) exposure on the model alga Chlamydomonas reinhardtii in the presence and absence of the essential micronutrient copper. The toxic ranges for Cu-NPs and the ionic control, CuCl2 , were determined using a high-throughput adenosine triphosphate (ATP)-based fluorescence assay. The Cu-NPs caused similar mortality in copper-replete and copper-deplete cells (median inhibitory concentration [IC50]: 14-16 mg/L) but were less toxic than the ionic control, CuCl2 (IC50: 7 mg/L). Using this concentration range, the Cu-NP impacts on cell morphology, copper accumulation, chlorophyll content, and expression of stress genes under both copper supply states were assessed. Osmotic swelling, membrane damage, and chloroplast and organelle disintegration were observed by transmission electron microscopy at both conditions. Despite these similarities, copper-deplete cells showed greater accumulation of loosely bound and tightly bound copper after exposure to Cu-NPs. Furthermore, copper-replete cells experienced greater loss of chlorophyll content, 19% for Cu-NPs, compared with only an 11% net decrease in copper-deplete cells. The tightly bound copper was bioavailable as assessed by reverse-transcriptase quantitative polymerase chain reaction analysis of CYC6, a biomarker for Cu deficiency. The increased resistance of copper-deplete cells to Cu-NPs suggests that these cells potentially metabolize excess Cu-NPs or better manage sudden influxes of ions. The results suggest that toxicity assessments must account for the nutritional status of impacted organisms and use toxicity models based on estimations of the bioavailable fractions.

Chlamydomonas reinhardtii cells exposed to abiotic stresses (e.g. nitrogen, zinc, or phosphorus deficiency) accumulate triacylglycerols (TAG), which are stored in lipid droplets. Here, we report that iron starvation leads to formation of lipid droplets and accumulation of TAGs. This occurs between 12 and 24 h after the switch to iron-starvation medium. C. reinhardtii cells deprived of iron have more saturated fatty acid (FA), possibly due to the loss of function of FA desaturases, which are iron-requiring enzymes with diiron centers. The abundance of a plastid acyl-ACP desaturase (FAB2) is decreased to the same degree as ferredoxin. Ferredoxin is a substrate of the desaturases and has been previously shown to be a major target of the iron deficiency response. The increase in saturated FA (C16:0 and C18:0) is concomitant with the decrease in unsaturated FA (C16:4, C18:3, or C18:4). This change was gradual for diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and digalactosyldiacylglycerol (DGDG), whereas the monogalactosyldiacylglycerol (MGDG) FA profile remained stable during the first 12 h, whereas MGDG levels were decreasing over the same period of time. These changes were detectable after only 2 h of iron starvation. On the other hand, DGTS and DGDG contents gradually decreased until a minimum was reached after 24-48 h. RNA-Seq analysis of iron-starved C. reinhardtii cells revealed notable changes in many transcripts coding for enzymes involved in FA metabolism. The mRNA abundances of genes coding for components involved in TAG accumulation (diacylglycerol acyltransferases or major lipid droplet protein) were increased. A more dramatic increase at the transcript level has been observed for many lipases, suggesting that major remodeling of lipid membranes occurs during iron starvation in C. reinhardtii.

We identified a Cu-accumulating structure with a dynamic role in intracellular Cu homeostasis. During Zn limitation, Chlamydomonas reinhardtii hyperaccumulates Cu, a process dependent on the nutritional Cu sensor CRR1, but it is functionally Cu deficient. Visualization of intracellular Cu revealed major Cu accumulation sites coincident with electron-dense structures that stained positive for low pH and polyphosphate, suggesting that they are lysosome-related organelles. Nano-secondary ion MS showed colocalization of Ca and Cu, and X-ray absorption spectroscopy was consistent with Cu(+) accumulation in an ordered structure. Zn resupply restored Cu homeostasis concomitant with reduced abundance of these structures. Cu isotope labeling demonstrated that sequestered Cu(+) became bioavailable for the synthesis of plastocyanin, and transcriptome profiling indicated that mobilized Cu became visible to CRR1. Cu trafficking to intracellular accumulation sites may be a strategy for preventing protein mismetallation during Zn deficiency and enabling efficient cuproprotein metallation or remetallation upon Zn resupply.